DIRECTED DIFFERENTIATION OF ES AND IPS CELLS TO HEMATOPOIETIC STEM CELLS

ES 和 IPS 细胞定向分化为造血干细胞

• 批准号: 8293183
• 负责人: KYUNGHEE CHOI
• 金额: $ 19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293183
• 关键词: Affect; Applications Grants; Basic Science; Biological Models; Biology; Blood; Blood Cells; Cell Lineage; Cell Transplants; Cells; Development; Disease; Embryo; Endothelial Cells; Endothelium; Engraftment; Frequencies; Funding Mechanisms; Generations; Genes; Goals; Growth; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Human Development; In Vitro; Individual; Medicine; Mesoderm; Molecular; Mus; Natural regeneration; Nature; Outcome; Pluripotent Stem Cells; Regenerative Medicine; SLAM protein; Somatic Cell; Sorting - Cell Movement; Stem cells; Testing; embryonic stem cell; improved; in vivo; induced pluripotent stem cell; novel; progenitor; reconstitution; regenerative; stem

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells provide an exciting opportunity in the field of regenerative biology and medicine, as they have the unique capacity to differentiate in culture into all somatic cells that make up an individual. Significant efforts have been made in generating functional hematopoietic stem cells (HSCs) from pluripotent stem cells. For instance, expression of HoxB4 (or Cdx4-HoxB4) during in vitro differentiation of ES cells has been shown to derive functional HSCs from ES cells (1-3). Despite these significant advancements, major challenges remain in utilizing in vitro derived HSCs and progenitors for regenerative medicine. For example, hematopoietic engraftment by ES derived hematopoietic progenitors remains consistently and disappointingly low. Even with HoxB4 expression, the frequency of HSCs and progenitors that can repopulate the recipient's hematopoietic system is estimated at best 1 in 2-5x106 among Cdx4-HoxB4 induced ES derived cells (3). Constitutive HoxB4 expression in transplanted cells could also be problematic. The overall objective is to robustly generate functional HSCs from ES and iPS cells. In achieving this goal, we posit that HSCs are generated from ES cells following the same molecular and cellular rules occurring in vivo. To this end, we demonstrated Flk-1 expressing (Flk-1+) hemangioblast, a common progenitor of hematopoietic and endothelial cells (or hemogenic endothelium), derived from in vitro differentiated ES cells can generate hematopoietic cells in culture (4, 5). In addition, we demonstrated that both primitive and definitive hematopoietic systems of the mouse are derived from the Flk-1+ mesoderm (6). Our preliminary studies indicate that when Flk-1+ hemangioblasts were sorted and further differentiated on OP9 cells, we were able to efficiently generate CD41+cKit+CD150+ cells, which have been previously identified as ES derived HSC/progenitors (3). Moreover, temporal co-expression of ER71, GATA2 and Scl positively induced CD41+cKit+CD150+ cells from ES cells: 1) by robustly inducing Flk-1+ hemangioblast formation from ES cells and 2) by independently inducing CD41+cKit+CD150+ cells from Flk-1+ hemangioblasts. Herein, we test a hypothesis that functional HSC generation from ES cells can be greatly improved by temporal ER71, GATA2 and Scl co-expression. We will determine if ES and iPS derived CD41+cKit+CD150+ cells could reconstitute hematopoietic system of the mouse and if temporal ER71, GATA2 and Scl co-expression would be superior to HoxB4 expression in generating functional HSCs. The ability to effectively direct ES and iPS cells to HSCs will provide a novel means of differentiation and will have a major potential impact in the field of regenerative biology and medicine. The outcome will also likely revolutionize the way we envision the cell lineage development and differentiation, thus will also have a major potential impact in the basic research field. Thus, the potential impact is high.

描述（由申请人提供）：胚胎茎（ES）细胞在再生生物学和医学领域提供了令人兴奋的机会，因为它们具有将培养物分化为组成个体的所有体细胞的独特能力。从多能干细胞产生功能性造血干细胞（HSC）方面已做出了重大努力。例如，在ES细胞的体外分化过程中，HOXB4（或CDX4-HOXB4）的表达已被证明从ES细胞中得出功能性HSC（1-3）。尽管取得了重大进步，但仍在利用体外衍生的HSC和祖细胞进行再生医学方面仍然存在重大挑战。例如，ES衍生的造血祖细胞的造血植入量保持一致且令人失望的低。即使使用HOXB4表达，在CDX4-HOXB4诱导的ES衍生细胞中，估计可以重新填充受体造血系统的HSC和祖细胞的频率在2-5x106中估计最多为2-5x106（3）。移植细胞中的组成型HOXB4表达也可能是有问题的。总体目标是从ES和IPS细胞中稳健地产生功能性HSC。在实现这一目标时，我们认为HSC是根据体内发生的相同分子和细胞规则从ES细胞产生的。为此，我们证明了FLK-1表达（FLK-1+）血管细胞，这是造血和内皮细胞的常见祖细胞（或血管内皮细胞），它源自体外分化ES细胞，可以在培养中产生血肿细胞（4，5）。此外，我们证明了小鼠的原始造血系统均来自FLK-1+中胚层（6）。我们的初步研究表明，当对FLK-1+血管细胞进行分类并在OP9细胞上进行进一步区分时，我们能够有效地产生CD41+ CKIT+ CD150+细胞，该细胞先前已被鉴定为ES衍生的HSC/祖细胞（3）。此外，通过鲁棒地诱导来自ES细胞的FLK-1+ Hemangioblast形成的ER71，GATA2和SCL的时间共表达CD41+ CKIT+ CKIT+ CD150+细胞：1）通过独立诱导来自FLK-1+ HemangioBlasts的CD41+ CKIT+ CKIT+ CKIT+ CKIT+ CKIT+ CKIT+ CKIT+ CCKIT+ CCKIT+ CCKIT+ CCKIT+ CCKIT+ CD150+细胞。在本文中，我们检验了一个假设，即颞ER71，GATA2和SCL共表达可以极大地改善ES细胞的功能性HSC生成。我们将确定ES和IPS得出的CD41+CKIT+CD150+细胞是否可以重建小鼠的造血系统，以及颞ER71，GATA2和SCL共表达是否优于HOXB4在生成功能性HSC中的HOXB4表达。有效地将ES和IPS细胞引导到HSC的能力将提供一种新颖的分化手段，并将对再生生物学和医学领域产生重大潜在影响。结果也可能会彻底改变我们设想细胞谱系发展和分化的方式，因此也将对基础研究领域产生重大潜在影响。因此，潜在影响很高。